Mammalian genomes constantly suffer from various types of damage, of which double-strand breaks (DSB) are considered the most dangerous (Haber, 2000). For example, DSBs can arise when the replication fork encounters a nick or when ionizing radiation particles create clusters of reactive oxygen species along their path. These reactive oxygen species may in turn themselves cause DSBs. For cultured mammalian cells that are dividing, 5-10% appear to have at least one chromosomal break (or chromatid gap) at any one time (Lieber & Karanjawala, 2004). Hence, the need to repair DSBs arises commonly (Li et al, 2007) and is critical for cell survival (Haber, 2000). Failure or incorrect repair can result in deleterious genomic rearrangements, cell cycle arrest, or cell death.
Repair of DSBs can occur through diverse mechanisms that can depend on cellular context. Repair via homologous recombination, the most accurate process, is able to restore the original sequence at the break. Because of its strict dependence on extensive sequence homology, this mechanism is suggested to be active mainly during the S and G2 phases of the cell cycle where the sister chromatids are in close proximity (Sonoda et al, 2006). Single-strand annealing is another homology-dependent process that can repair DSB between direct repeats and thereby promotes deletions (Paques & Haber, 1999). Finally, non-homologous end joining (NHEJ) of DNA is a major pathway for the repair of DSBs because it can function throughout the cell cycle and because it does not require a homologous chromosome (Moore & Haber, 1996).
NHEJ comprises at least two different processes (Feldmann et al, 2000). The main and best characterized mechanism involves rejoining of what remains of the two DNA ends through direct re-ligation (Critchlow & Jackson, 1998) or via the so-called microhomology-mediated end joining (MMEJ) (Ma et al, 2003). Although perfect re-ligation of the broken ends is probably the most frequent event, it could be accompanied by the loss or gain of several nucleotides.
Like most DNA repair processes, there are three enzymatic activities required for repair of DSBs by the NHEJ pathway: (i) nucleases to remove damaged DNA; (ii) polymerases to aid in the repair, and; (iii) a ligase to restore the phosphodiester backbone. Depending on the nature of the DNA ends, DNA can be simply re-ligated or terminal nucleotides can be modified or removed by inherent enzymatic activities, such as phosphokinases and exonucleases. Missing nucleotides can also be added by polymerase μ or λ. In addition, an alternative or so-called back-up pathway has been described that does not depend on ligase IV and Ku components and has been involved in class switch and V(D)J recombination (Ma et al, 2003). Overall, NHEJ can be viewed as a flexible pathway wherein the goal is to restore the chromosomal integrity, even at the cost of nucleotide excisions or insertions.
DNA repair can be triggered by both physical and chemical means. Several chemicals are known to cause DNA lesions and are used routinely. Radiomimetic agents, for example, work through free-radical attack on the sugar moieties of DNA (Povirk, 1996). A second group of drugs inducing DNA damage includes inhibitors of topoisomerase I (TopoI) and II (TopoII) (Burden & N., 1998; Teicher, 2008). Other classes of chemicals bind covalently to the DNA and form bulky adducts that are repaired by the nucleotide excision repair (NER) system (Nouspikel, 2009). Chemicals inducing DNA damage have a diverse range of applications, however, although certain agents are more commonly applied in studying a particular repair pathway (e.g. cross-linking agents are favored for NER studies), most drugs simultaneously provoke a variety of lesions (Nagy & Soutoglou, 2009). Furthermore, using these classical strategies the overall yield of induced mutations is quite low, and the DNA damage leading to mutagenesis cannot be targeted to precise genomic DNA sequence.
The most widely used site-directed mutagenesis strategy is gene targeting (GT) via homologous recombination (HR). Efficient GT procedures in yeast and mouse have been available for more than 20 years (Capecchi, 1989; Rothstein, 1991). Successful GT has also been achieved in Arabidopsis and rice plants (Endo et al, 2006; Endo et al, 2007; Hanin et al, 2001; Terada et al, 2002). Typically, GT events occur in a fairly small population of treated mammalian cells and are extremely low in higher plant cells, in the range of 0.01-0.1% of the total number of random integration events (Terada et al, 2007). The low GT frequencies reported in various organisms are thought to result from competition between HR and NHEJ for repair of DSBs. There are extensive data indicating that DSB repair by NHEJ is error-prone due to end-joining processes that generate insertions and/or deletions (Britt, 1999). Thus, these NHEJ-based strategies might be more effective than HR-based strategies for targeted mutagenesis into cells.
Expression of I-SceI, a rare cutting endonuclease, has been shown to introduce mutations at I-SceI cleavage sites in mammalian cells (Liang et al, 1998), Arabidopsis and tobacco (Endo et al, 2006; Endo et al, 2007; Hanin et al, 2001; Kirik et al, 2000; Terada et al, 2007). However, the use of endonucleases is limited to rarely occurring natural recognition sites or to artificially introduced target sites. To overcome this problem, meganucleases with engineered specificity towards a chosen sequence have been developed (Arnould et al, 2006a; Arnould et al, 2007; Grizot et al, 2009; Smith et al, 2006). Meganucleases show high specificity to their DNA target, these proteins being able to cleave a unique chromosomal sequence and therefore do not affect global genome integrity. Natural meganucleases are essentially represented by homing endonucleases, a widespread class of proteins found in eukaryotes, bacteria and archae (Chevalier & Stoddard, 2001). Homing endonucleases can be classified in five different families, the largest and best characterized one being the LAGLIDAG homing endonuclease family, named after a conserved sequence motif (Stoddard, 2005). LAGLIDADG homing endonucleases can be dimeric (possessing a single LAGLIDADG motif per polypeptide), or monomeric (possessing two LAGLIDADG motifs per polypeptide).
Early studies of the I-SceI and HO homing endonucleases illustrated how the cleavage activities of these proteins could be used to initiate HR events in living cells and demonstrated the recombinogenic properties of chromosomal DSBs (Dujon et al, 1986; Haber, 1995). Since then, I-SceI-induced HR has been successfully used for genome engineering purposes in bacteria (Posfai et al, 1999b), mammalian cells (Cohen-Tannoudji et al, 1998; Donoho et al, 1998; Grizot et al, 2009; Sargent et al, 1997), mice (Gouble et al, 2006) and plants (Puchta et al, 1996; Siebert & Puchta, 2002). Meganucleases have emerged as the scaffolds of choice for deriving genome engineering tools cutting a desired target sequence (Paques & Duchateau, 2007b). Combinatorial assembly processes allowing for the engineering of meganucleases with modified specificities have been described (Arnould et al, 2006a; Arnould et al, 2007; Grizot et al, 2009; Smith et al, 2006). Briefly, these processes rely on the identification of locally engineered variants with a substrate specificity that differs from that of the wild-type meganuclease by only a few nucleotides.
Zinc-finger nucleases (ZFNs) represent another type of specific nuclease. ZFNs are chimeric proteins composed of a synthetic zinc-finger-based DNA binding domain fused to a DNA cleavage domain. By modification of the zinc-finger DNA binding domain, ZFNs can be specifically designed to cleave virtually any long stretch of dsDNA sequence (Cathomen & Joung, 2008; Kim et al, 1996). A NHEJ-based targeted mutagenesis strategy was recently developed for several organisms by using synthetic ZFNs to generate DSBs at specific genomic sites (Beumer et al, 2008; Doyon et al, 2008a; Holt et al, 2010; Lloyd et al, 2005; Meng et al, 2008; Perez et al, 2008; Santiago et al, 2008). Subsequent repair of the DSBs by NHEJ frequently produces deletions and/or insertions at the joining site. For example, in zebrafish embryos the injection of mRNA coding for engineered ZFNs led to animals carrying the desired heritable mutations (Doyon et al, 2008b). In plant, similar NHEJ-based targeted mutagenesis has also been successfully applied (Lloyd et al, 2005). Although these powerful tools are available, there is still a need to further improve double-strand break-induced mutagenesis.
Recent studies suggest that co-expressing TREX2 exonuclease with I-SceI meganuclease modify gene repair activity in mouse ES cells, thereby causing partial degradation of chromosomal DNA by I-SceI (Bennardo et al, 2009). However, it was found in these studies that limiting the persistence of a chromosome break with TREX 2, diminishes the mutagenic potential of the meganuclease.
In this context, the inventors have developed an original approach to increase the efficiency of targeted DSB-induced mutagenesis based on the use of single-chain proteins combining TREX2 exonuclease with different rare-cutting endonucleases, such LAGLIDADG homing endonucleases and TALENs.
This approach has proven particular efficiency for gene mutagenesis, especially in plant transformation.